Cells interact with different types of extracellular matrix in different tissues and at different stages of embryonic development and adult wound repair. This project focuses on addressing the following major questions concerning the mechanisms of these cell-extracellular matrix interactions:[unreadable] 1. How do cells assemble a three-dimensional (3D) extracellular matrix, particularly one that is primarily based on fibronectin fibrils?[unreadable] 2. What are the differences between signaling and biology in traditional two-dimensional (2D) cell culture and various 3D matrices characteristic of in vivo microenvironments?[unreadable] 3. What unique mechanisms control cell behavior in 3D microenvironments?[unreadable] [unreadable] We previously analyzed the molecular machinery mediating integrin-based assembly of a 3D fibronectin matrix and published evidence for the role of integrin activation and concerted protein translocation to generate fibronectin fibrils. We are continuing to characterize the regulation of matrix assembly, signal transduction, and cytoskeletal mechanisms using a set of novel integrin mutants we have generated that display specific defects in fibronectin matrix assembly. Our recent preliminary findings suggest that fibronectin matrix assembly depends on multiple sites in the integrin cytoplasmic domain. Depending on the point mutation, matrix assembly can be disrupted differentially compared to integrin-mediated adhesion.[unreadable] [unreadable] We previously published evidence for the importance of the three-dimensionality of extracellular matrix surrounding fibroblasts in a variety of cell biological functions including migration and proliferation. Very recently, we completed a review of the field of 3D models of tissue morphogenesis and cancer. Based on the current literature, we have initiated tests of two new hypotheses concerning the mechanisms of cell migration in 3D settings. Cells are known to be surrounded by biochemically and structurally distinct matrices in vivo, e.g., a matrix rich in fibronectin fibrils during early craniofacial neural crest migration versus a collagen-rich matrix with varying crosslinking in adult connective tissue. We hypothesize that (1) three-dimensionality, and differences in biochemical composition or crosslinking can combine to determine altered requirements for cytoskeletal and proteolytic responses needed for cell migration. We further hypothesize that (2) the role of myosin IIA (and possibly IIB) may differ in 3D versus 2D settings, as well as in different types of 3D environments. Our preliminary data suggest that depending on the type of 3D matrix (cell-derived versus collagen gels), myosin II isoforms may or may not be required for effective cell migration; this dichotomy in 3D contrasts with its migratory down-regulatory function in 2D cell culture, where its ablation stimulates migration. We will test for further differences in 3D environments, compare requirements in other types of 3D matrices, and determine whether they can be explained by distinct interactions and intracellular regulation due to the use of different integrins and subunits, e.g., the collagen-binding alpha-2 subunit versus the fibronectin-binding alpha-5 integrin.[unreadable] [unreadable] We are also developing methods to visualize cell-surface and cytoskeletal dynamics in 3D matrices. Although technically difficult, this microscopy technology will be important to develop further to permit direct comparisons of cellular functions in 2D versus 3D environments. Understanding whether the extent and nature of requirements for a specific protein differ in 2D and 3D appears important, since initial conclusions from in 2D vitro studies may differ under 3D or in vivo conditions.